Kita, Hirohito

Abstract Description Early oral route antigen exposure decreases the incidence of food allergy while environmental exposures through non-oral routes, such as the skin or airways, increase the risk. The goal of this study was to investigate the immunologic mechanisms of how oral allergen consumption protects from sensitization to the same allergens through environmental exposure routes. Naive mice were orally administered ovalbumin (OVA) in drinking water. Mice were subsequently exposed intranasally (i.n.) or epicutaneously (e.c.) to OVA plus peanut flour, as an adjuvant. OVA-specific CD4+ T cells from draining lymph nodes (dLNs) were assessed by MHC-II tetramer staining and flow cytometry. Following i.n. or e.c. exposure to OVA plus peanut flour, mice developed OVA-IgE and OVA-IgG1 and showed symptoms of acute anaphylaxis upon OVA challenge. Oral OVA feeding prior to i.n. or e.c. antigen exposure dramatically reduced OVA-IgE titers and protected mice from anaphylaxis. Protection was OVA-specific, as OVA-fed mice still developed peanut-IgE and anaphylaxis upon i.p. peanut challenge. Total OVA-specific CD4+ T and CD4+Foxp3+ T regulatory (Treg) cell numbers in dLNs were similar between control and OVA-fed mice. However, the majority of OVA-specific CD4+ T cells in control dLNs were T follicular helper (Tfh) cells, which were nearly abolished in OVA-fed mice. Oral route antigen exposure suppresses development of antigen-specific Tfh cells in remote dLNs and prevents systemic allergic sensitization. Funding Sources Supported by NIH R37 AI71106 and Mayo Clinic Graduate School of Biomedical Sciences. Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Abstract Description The immunological mechanism involved in regulation of chronic airway inflammation in allergic asthma is not fully understood. This study aimed to identify subpopulation(s) of T regulatory (Treg) cell responsible for controlling type 2 airway inflammation. Treg cells in the lung tissues of fungal allergen Alternaria-exposed mice expressed higher levels of Il2ra, Pdcd1, Ctla4, Il10 as compared to those in mediastinal lymph nodes. Mass cytometry analysis identified several populations of Treg cells including the one highly expressed CCR8, ST2, and GATA-3. CCR8+ST2+ Treg cells expanded after 4-week of Alternaria exposure and persisted in the lung tissues for additional 8 weeks without allergen re-exposure; their tissue residency was not affected by FTY720 treatment. Transcriptional analysis showed that CCR8+ST2+ Treg cells were enriched for Gata3 while minimally expressed type 2 cytokines. Moreover, CCR8+ST2+ Treg cells produced higher levels of IL-10 and amphiregulin compared to CCR8-ST2+ Treg cells and effectively suppressed proliferation of effector T cells. Severe airway inflammation and asthma-like pathology developed in Treg cell-deficient (Foxp3DTR) and Ccr8-/- mice underscored the functional significance of CCR8+ST2+ Treg cells to control airway inflammation in vivo. These findings suggest that heterogenous populations of Treg cells develop in the lung tissues during chronic airway inflammation and CCR8+ST2+ Treg subpopulation is likely to be highly suppressive. Funding Sources Supported by NIH grant R37 AI71106; Mayo Foundation; Mayo Graduate School of Biomedical Sciences Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Background Eosinophilic oesophagitis (EoE) is a food allergen-induced inflammatory disorder characterised by interleukin (IL)-13-mediated oesophageal inflammation and epithelial basal cell hyperplasia (BCH). The role of mitochondria in EoE pathogenesis remains elusive. Design Prompted by single cell transcriptomics data, we interrogated the role of mitochondria in EoE pathobiology using patient biopsies, EoE-mouse models and oesophageal epithelial cells grown in monolayer and three-dimensional (3D) organoid cultures treated with EoE-relevant cytokines. 3D organoids and EoE-bearing mice were treated with omeprazole—a proton-pump inhibitor used as first-line EoE therapy. We performed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference in mouse organoids to identify the key mitochondrial regulatory genes whose depletion may lead to BCH. We analysed mitochondrial membrane potential, mass and superoxide production by flow cytometry, cellular oxygen consumption by respirometry, mitochondrial structures and perturbation of cellular energy homeostasis by immunoblotting. Results Mitochondrial dysfunction appeared to be a hallmark of EoE-related BCH where mitochondrial structural damage was associated with impaired oxidative respiratory capacity, elevation of mitochondrial superoxide and decreased adenosine triphosphate (ATP) production, as corroborated by activation of the adenosine monophosphate (AMP) -activated protein kinase and suppression of mammalian target-of-rapamycin signalling. Depletion of PGC1A, the master regulator of mitochondria biogenesis, recapitulated EoE-related BCH, suggesting that mitochondrial dysfunction drives BCH. Further, omeprazole alleviated mitochondrial damage and dysfunction in EoE-related BCH modelled in mice and patient-derived organoids. Conclusion Mitochondrial dysfunction is tightly linked to perturbation of redox homeostasis in EoE-related BCH, which is promoted by IL-13 and reversible with omeprazole treatment.

Allergens from the fungus Alternaria alternata promote secretion of alarmins from the airway epithelium including ATP, IL-33 and both genomic and mitochondrial DNA fragments, which produce type 2 immune responses in airways. Alternaria -induced oxidative stress activates ATP release by opening apical voltage-dependent anion channels (VDAC-1) and stimulating exocytosis of ATP. This investigation demonstrated that treatment of human bronchial epithelial (hBE) cells with a selective peptide antagonist or small molecule inhibitor (FPS-ZM 1 ) of the receptor for advanced glycation end products (RAGE), reduced Alternaria -evoked ATP secretion, decreasing the initial rate by ~40%. Furthermore, the Alternaria -induced increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) was inhibited by ~75% following pretreatment with RAGE antagonist peptide. Intranasal administration of Alternaria extract (50 μg) produced a significant increase in IL-33 secretion into bronchoalveolar lavage (BAL) fluid, which was inhibited in RAGE-deficient ( Ager -/- ) mice. Moreover, Alternaria -induced increases in IL-5 and IL-13 secretion into BAL fluid were also significantly reduced in Ager -/- mice. These findings support the conclusion that Alternaria -dependent ATP release and Ca 2+ mobilization was dependent in part on RAGE activation and that RAGE antagonists have the potential of reducing type 2 immune responses in airways following Alternaria allergen exposure. This study was supported by grants from the NIH (R01A1128729 & 1R01AI169530) to HK and SMO and by the Minnesota Agricultural Experiment station grant (AES0016134) to SMO. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Advanced‐stage liver cancers are associated with poor prognosis and have limited treatment options, often leading the patient to hospice care. Percutaneous intratumoral injection of anticancer agents has emerged as a potential alternative to systemic therapy to overcome tumor barriers, increase bioavailability, potentiate immunotherapy, and avoid systemic toxicity, which advanced‐stage cancer patients cannot tolerate. Here, an injectable OncoGel (OG) comprising of a nanocomposite hydrogel loaded with an ionic liquid (IL) is developed for achieving a predictable and uniform tumor ablation and long‐term slow release of anticancer agents into the ablation zone. Rigorous mechanical, physiochemical, drug release, cytotoxicity experiments, and ex vivo human tissue testing identify an injectable version of the OG with bactericidal properties against highly resistant bacteria. Intratumoral injection of OG loaded with Nivolumab (Nivo) and doxorubicin (Dox) into highly malignant tumor models in mice, rats, and rabbits demonstrates enhanced survival and tumor regression associated with robust tissue ablation and drug distribution throughout the tumor. Mass cytometry and proteomic studies in a mouse model of colorectal cancer that often metastasizes to the liver indicate an enhanced anticancer immune response following the intratumoral injection of OG. OG may augment immunotherapy and potentially improve outcomes in liver cancer patients.

Allergens derived from the fungus Alternaria alternata stimulate the release of multiple alarmins including ATP, IL-33 and DNA fragments from human bronchial epithelial cells (hBECs). Oxidative stress induced by Alternaria extract activates ATP release by opening voltage-dependent anion channels (VDAC-1) in the apical membrane and by stimulating exocytosis of ATP containing membrane vesicles. The present study shows that pretreatment of hBECs with K2P channel inhibitors prior to Alternaria exposure significantly increases the initial rate of ATP release and inhibition of ATP loading into membrane vesicles with bafilomycin A1 blocks the response. Furthermore, pretreatment with the K+ channel openers, EBIO (KCa3.1 channels), NS11021 (KCa1.1 channels) and pinacidil (KATP channels) each inhibited the bafilomycin A1-dependent component of ATP release, reducing the initial rate of ATP secretion by ~50%. K2P channel blockers caused depolarization whereas K+ channel openers produced hyperpolarization of the cell membrane. Additionally, K2P channel inhibitors also increased Alternaria-stimulated Ca2+ uptake into hBECs while K+ channel openers reduced the Alternaria response by ~50%. Imaging experiments involving visualization of fluorescently labeled MARCKS proteins in the apical membrane showed that treatment with Alternaria caused a significant decrease in fluorescence intensity, however pretreatment with K+ channel openers, but not K2P channel blockers, inhibited the effect. These results are consistent with recent findings showing that MARCKS proteins bind to the plasma membrane through an electrostatic association with PIP2 and that phosphorylation by PKC or membrane hyperpolarization following K+ channel activation disrupts this interaction resulting in MARCKS detachment from the membrane. MARCKS detachment uncouples the membrane from the actin cytoskeleton, facilitating vesicle docking, fusion and release of ATP. This study was supported by NIH grants (AI128729 and AI169530) to HK and SMO and by USDA-AES 0016134 to SMO. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Environmental allergens that interact with the airway epithelium can activate cellular stress pathways that lead to the release of danger signals known as alarmins. The mechanisms of alarmin release are distinct from damage-associated molecular patterns (DAMPs), which typically escape from cells after loss of plasma membrane integrity. Oxidative stress represents a form of allergen-induced cellular stress that stimulates oxidant-sensing mechanisms coupled to pathways, which facilitate alarmin mobilization and efflux across the plasma membrane. In this review, we highlight examples of alarmin release and discuss their roles in the initiation of type 2 immunity and allergic airway inflammation. In addition, we discuss the concept of alarmin amplification, where “primary” alarmins, which are directly released in response to a specific cellular stress, stimulate additional signaling pathways that lead to secretion of “secondary” alarmins that include proinflammatory cytokines, such as IL-33, as well as genomic and mitochondrial DNA that coordinate or amplify type 2 immunity. Accordingly, allergen-evoked cellular stress can elicit a hierarchy of alarmin signaling responses from the airway epithelium that trigger local innate immune reactions, impact adaptive immunity, and exacerbate diseases including asthma and other chronic inflammatory conditions that affect airway function.

Background Epigenetics studies mechanisms such as DNA methylation, histone modifications, non-coding RNAs, and alternative polyadenylation that can modify gene activity without changing the underlying DNA nucleotide base-pair structure. Because these changes are reversible, they have potential in developing novel therapeutics. Currently, seven pharmaceutical agents targeting epigenetic changes are FDA approved and commercially available for treatment of certain cancers. However, studies investigating epigenetics in chronic rhinosinusitis (CRS) have not been undertaken previously in the United States. Objectives The goal of this study was to investigate sinonasal DNA methylation patterns in CRS versus controls, to discern environmentally-induced epigenetic changes impacting CRS subjects. Methods and Results Ethmoidal samples from CRS and inferior turbinate mucosal tissue samples from controls without CRS were studied. DNA methylation was studied by Reduced Representation Bisulfite Sequencing. RADMeth® biostatistical package was used to identify differentially methylated regions (DMRs) between CRS and controls. Ingenuity Pathway analysis of DMRs was performed to identify top upstream regulators and canonical pathways. Ninety-three samples from 64 CRS subjects (36 CRSwNP; 28 CRSsNP) and 29 controls were studied. CRS and control samples differed in 13 662 CpGs sites and 1381 DMRs. Top upstream regulators identified included: 1. CRS versus controls: TGFB1, TNF, TP53, DGCR8, and beta-estradiol. 2. CRSwNP versus controls: TGFB1, CTNNB1, lipopolysaccharide, ID2, and TCF7L2. 3. CRSsNP versus controls: MYOD1, acetone, ID2, ST8SIA4, and LEPR. Conclusions Differential patterns of methylation were identified between controls and CRS, CRSwNP, and CRSsNP. Epigenetic, environmentally-induced changes related to novel, inflammatory, immunologic, and remodeling pathways appear to affect epithelial integrity, cell proliferation, homeostasis, vascular permeability, and other yet uncharacterized pathways and genes.